Centro di Biologia Integrata - CIBIO , Università di Trento , 38123 Povo , Trento , Italy.
Dipartimento di Scienze degli Alimenti e del Farmaco , Università di Parma , 43124 Parma , Italy.
J Phys Chem B. 2018 Dec 13;122(49):11326-11337. doi: 10.1021/acs.jpcb.8b07260. Epub 2018 Sep 17.
The fluorescence of Green Fluorescent Protein (wtGFP) and variants has been exploited in distinct applications in cellular and analytical biology. GFPs emission depends on the population of the protonated (A-state) and deprotonated (B-state) forms of the chromophore. Whereas wtGFP is pH-independent, mutants in which Ser65 is replaced by either threonine or alanine (as in GFPmut2) are pH-dependent, with a p K around 6. Given the wtGFP pH-independence, only the structure of the protonated form was determined. The deprotonated form was deduced on the basis of the crystal structure of the Ser65Thr mutant at basic pH, assuming that it corresponds to the conformation populated in solution. Here, we present an investigation where structures of the protonated and deprotonated forms of GFPmut2 were determined from crystals grown in either MPD at pH 6 or PEG at pH 8.5, and moved to either higher or lower pH. Both crystal forms of GFPmut2 were titrated monitoring the process via polarized absorption microspectrophotometry in order to precisely correlate the protonation process with the structures. We found that (i) in solution, chromophore titration is not thermodynamically coupled with any residue and Glu222 is always protonated independent of the protonation state of the chromophore; (ii) the lack of coupling is reflected in the structural behavior of the chromophore and Glu222 environments, with only the former showing variations with pH; (iii) titrations of low-pH and high-pH grown crystals exhibit a Hill coefficient of about 0.75, indicating an anticooperative behavior not observed in solution; (iv) structures where pH was changed in the crystal point to Glu222 as the ionizable group responsible for the outset of the anticooperative behavior; and (v) in GFPmut2 the canonical GFP proton wire involving the chromophore is not interrupted at the level of Ser205 and Glu222 at basic pH as in the Ser65Thr mutant. This allows proposing the structure of the deprotonated state of GFPmut2 as an alternative model for the analogous state of wtGFP.
绿色荧光蛋白（wtGFP）及其变体的荧光已被广泛应用于细胞和分析生物学中的不同应用。GFP 的发射取决于发色团质子化（A 态）和去质子化（B 态）形式的种群。虽然 wtGFP 是 pH 独立的，但 Ser65 被苏氨酸或丙氨酸取代的突变体（如 GFPmut2）是 pH 依赖的，pK 值约为 6。鉴于 wtGFP 的 pH 独立性，仅确定了质子化形式的结构。根据 Ser65Thr 突变体在碱性 pH 下的晶体结构，推断出去质子化形式，假设它对应于溶液中存在的构象。在这里，我们介绍了一项研究，其中确定了 GFPmut2 的质子化和去质子化形式的结构，这些结构是从在 pH 6 的 MPD 或 pH 8.5 的 PEG 中生长的晶体中获得的，并转移到更高或更低的 pH 值。 GFPmut2 的两种晶体形式都通过偏振吸收微分光光度法进行滴定监测，以精确地将质子化过程与结构相关联。我们发现：（i）在溶液中，发色团滴定与任何残基没有热力学偶联，并且 Glu222 始终是质子化的，与发色团的质子化状态无关；（ii）缺乏偶联反映在发色团和 Glu222 环境的结构行为中，只有前者随 pH 值而变化；（iii）低 pH 和高 pH 生长晶体的滴定显示出约 0.75 的希尔系数，表明在溶液中未观察到的反协同行为；（iv）在晶体中改变 pH 值的结构指向 Glu222 作为负责反协同行为开始的可电离基团；（v）在 GFPmut2 中，涉及发色团的经典 GFP 质子线在 Ser205 和 Glu222 处不会像 Ser65Thr 突变体那样在碱性 pH 下中断。这允许将 GFPmut2 的去质子化状态的结构作为 wtGFP 的类似状态的替代模型。
